RNA interference (RNAi) is a near-ubiquitous pathway involved in post-transcriptional gene modulation. A key effector molecule of RNAi is the microRNA (miRNA or miR). These small, non-coding RNAs are transcribed as primary miRNAs (pri-miRNA) and processed in the nucleus by Drosha (a Type III ribonuclease) to generate short hairpin structures referred to as pre-miRNAs (FIG. 1). The resulting molecules are transported to the cytoplasm and processed by a second nuclease (Dicer) before being incorporated into the RNA Induced Silencing Complex (RISC). Interactions between the mature miRNA-RISC complex and messenger RNA (mRNA), particularly between the seed region of the miRNA guide strand (nucleotides 2-7) and regions of the 3′ UTR of the mRNA, leads to gene knockdown by transcript cleavage and/or translation attenuation.
While study of native substrates (miRNA) has garnered considerable interest in recent years, the RNAi pathway has also been recognized as a powerful research tool. Small double stranded RNAs (referred to as small interfering RNAs or siRNA) generated by synthetic chemistries or enzymatic methods can be introduced into cells by a variety of means (e.g. lipid mediated transfection, electroporation) and enter the pathway to target specific gene transcripts for degradation. As such, the RNAi pathway serves as a potent tool in the investigation of gene function, pathway analysis, and drug discovery, and is envisioned to have future applications as a therapeutic agent.
Though the use of synthetic siRNA serves the needs of most gene knockdown experiments, there are some instances where synthetic molecules are unsuitable. A fraction of the cell types are resilient or highly sensitive to commonly used transfection methods and/or reagents. In still other instances, the needs of the experimental system require that gene knockdown be achieved for periods longer than those provided by synthetic molecules (typically 4-10 days).
Vector-based delivery of silencing reagents has previously been achieved using a range of delivery (e.g., lentiviral) and scaffold (simple hairpins, miRNA-based) configurations (Samakoglu et al., Nature Biotech.; Lei Y. S. et al., 2005; Leirdal and Sioud, 2002; Anderson et al., 2003; Grimm, D. et al., (2006) Nature Letters 441:537-541).